Mycobacterium tuberculosis accounts for more deaths worldwide than any other infectious agent. Recent increases in the number of cases have been associated with the spread of the AIDS virus and the appearance of drug-resistant strains of M. tuberculosis. The development of new treatments for mycobacteria requires an understanding of the biology of these bacteria and the ability to manipulate their genomes to determine the genetic basis of pathogenesis and drug resistance. In M. tuberculosis, studies have been hindered by inefficient methods for the introduction of DNA into and between strains, and by the low frequency of targeted recombination. In particular, the ability to make site-directed changes within the M. tuberculosis genome is crucial for understanding the role of particular genes in virulence. We propose to characterize a novel conjugation system that can transfer chromosomal DNA between strains of Mycobacterium smegmatis and use it as a tool for the analysis and transfer of DNA between mycobacteria. This naturally occurring system could offer an efficient method for the introduction of DNA into the slow-growing pathogenic mycobacteria. Furthermore, by examining conjugation between different species of mycobacteria we will assess its promiscuity and possible role in the spread of drug resistance. Finally, we have identified three M. tuberculosis genes that are highly homologous to those encoding the RecBCD pathway of recombination in Escherichia coli, which is the major pathway for conjugal recombination and double-strand break repair. We intend to characterize these genes to facilitate the development of a defined homologous recombination system in M. tuberculosis. These goals will be achieved by: 1. Cloning and characterizing the cis-acting sequences necessary for conjugal DNA transfer. 2. Identifying the trans-acting protein functions necessary for transfer. 3. Monitoring conjugal transfer from M. smegmatis into other mycobacterial species including the slow-growing pathogenic species. 4. Developing a defined allele-exchange system by characterizing the RecBCD pathway of M. tuberculosis